The KRASG12C Inhibitor MRTX849 Reconditions the Tumor Immune Microenvironment and Sensitizes Tumors to Checkpoint Inhibitor Therapy

Abstract

KRAS^G12C inhibitors, including MRTX849, are promising treatment options for KRAS-mutant non-small cell lung cancer (NSCLC). PD-1 inhibitors are approved in NSCLC; however, strategies to enhance checkpoint inhibitor therapy (CIT) are needed. KRAS^G12C mutations are smoking-associated transversion mutations associated with high tumor mutation burden, PD-L1 positivity, and an immunosuppressive tumor microenvironment. To evaluate the potential of MRTX849 to augment CIT, its impact on immune signaling and response to CIT was evaluated. In human tumor xenograft models, MRTX849 increased MHC class I protein expression and decreased RNA and/or plasma protein levels of immunosuppressive factors. In a Kras^G12C -mutant CT26 syngeneic mouse model, MRTX849 decreased intratumoral myeloid-derived suppressor cells and increased M1-polarized macrophages, dendritic cells, CD4⁺, and CD8⁺ T cells. Similar results were observed in lung Kras^G12C -mutant syngeneic and a genetically engineered mouse (GEM) model. In the CT26 Kras^G12C model, MRTX849 demonstrated marked tumor regression when tumors were established in immune-competent BALB/c mice; however, the effect was diminished when tumors were grown in T-cell-deficient nu/nu mice. Tumors progressed following anti-PD-1 or MRTX849 single-agent treatment in immune-competent mice; however, combination treatment demonstrated durable, complete responses (CRs). Tumors did not reestablish in the same mice that exhibited durable CRs when rechallenged with tumor cell inoculum, demonstrating these mice developed adaptive antitumor immunity. In a GEM model, treatment with MRTX849 plus anti-PD-1 led to increased progression-free survival compared with either single agent alone. These data demonstrate KRAS inhibition reverses an immunosuppressive tumor microenvironment and sensitizes tumors to CIT through multiple mechanisms.

Figure 1.

KRAS^G12C regulates antigen presentation and an immunosuppressive tumor microenvironment in a tumor cell–intrinsic manner. A, Heatmap of log₂ fold changes in RNA expression from RNA-seq data of selected immune-modulating genes in human tumor xenografts established in immune-compromised mice dosed orally with MRTX849 once every day, five times per day or seven times per day and harvested 6 or 24 hours after administration relative to expression in vehicle-treated tumors. “*” indicates FDR < 0.05, n = 3 per treatment group. B, Plasma concentration of selected secreted immune-modulating proteins in immune-compromised mice harboring human tumor xenografts orally administered vehicle or MRTX849 every day or for the number of days indicated. Plasma was collected 24 hours after last dose for the H1373 and H358 models and as indicated for other models. “*” indicates Student t test, P = 0.05, MRTX849-treated plasma compared with vehicle-treated plasma, n = 3 per treatment group. Veh = vehicle; Pre Tx = pretreatment. C, log₂ expression of human MHC class I protein in human xenograft models established in immune-compromisedmice orallyadministered vehicle or 100 mg/kg MRTX849 once every day, five times per day or seven times per day. Presence of a bar indicates statistically significant difference between indicated groups by Student t test, P < 0.05, n = 3 per treatment group. Two different vehicle-treated tumor volume groups (300 and 500 mm³) were used for the H358 study.

Figure 2.

CRISPR/Cas9-engineered CT26 Kras^G12C cells and tumors are sensitive to MRTX849 treatment. A, Parental CT26.WT and G12C-engineered CT26.WT cell lines (CT26 Kras^G12C) derived from single-cell clones (clone E3 and F6) were incubated with 300 nmol/L of MRTX849 for 24 hours and probed via immunoblot with total anti-KRAS and anti-α-tubulin antibodies. The increased molecular weight of KRAS^G12C in the MRTX849-treated lane is indicative of covalent KRAS^G12C modification. The molecular weight of the standard protein to the left of the DMSO-treated parental lysate is 20 kD. B, A Cell Titer-Glo viability assay was performed on parental and CT26 Kras^G12C E3 and F6 cell line clones treated with MRTX849 in vitro for 3 days. C, Parental CT26.WT and CT26 Kras^G12C clone E3 cells were incubated with 1,000 nmol/L of MRTX849 for 6 or 24 hours and lysates were probed via immunoblot with anti-KRAS, pERK (Thr202/Tyr204), ERK, pS6 (Ser235/236), S6 or α-tubulin. D, 100 mg/kg MRTX849 was administered orally to mice bearing CT26 Kras^G12C E3 tumors. Tumors were harvested 6 hours after dosing and lysates were probed via immunoblot with anti-KRAS, pERK (Thr202/Tyr204), ERK, pS6 (Ser235/236), S6 or α-tubulin. E, 10, 30, and 100 mg/kg MRTX849 was administered orally every day to BALB/cmice bearing established CT26 Kras^G12C tumors (n = 7 per treatment group; average starting tumor volume ~100mm³) and tumor volume was measured over the study at indicated days. Average tumor volume ± SEM shown.

Figure 3.

MRTX849 and combined MRTX849 and anti–PD-1 administration markedly alters immune cell populations in the tumor microenvironment. MRTX849 (100 mg/kg), rat IgG2a isotype Control antibody (10 mg/kg, Bio X cell lot No. 686318F1B), and mouse PD-1 antibody at 10 mg/kg (Bio X cell clone 29F.1A12) were administered orally, daily (MRTX849) or intraperitoneally, every 3 days (PD-1 and isotype control) or in combination to mice bearing established, subcutaneous CT26 Kras^G12C tumors (n = 4 per treatment group; average starting tumor volume approximately 220 mm³ (vehicle and PD-1–treated cohorts) or approximately 730 mm₃ (MRTX849 or combination-treated cohorts. Intratumoral myeloid (A) and lymphoid (B) populations were analyzed in excised disaggregated tumors by flow cytometry following 4 or 8 days of treatment administration. Analyzed cell populations include DC—dendritic cell, M1MAC—M1-polarized macrophage, M2MAC—M2-polarized macrophage, G-MDSC—granulocytic myeloid-derived suppressor cell, M-MDSC—monocytic MDSC, Tregs—T regulatory cells, NK T—natural killer T cells denoted by cell lineage–specific markers including CD11b, CD45, CD3, CD4, CD8, etc. (see Supplementary Methods). Flow cytometry analysis on single-cell suspensions of CT26 KRAS^G12C tumors treated with vehicle or MRTX849 was run twice and data were similar between experiments. Bars denotes statistical significance by one-way ANOVA with post hoc Tukey test.

Figure 4.

MRTX849 and anti–PD-1 combination treatment leads to durable complete responses in the majority of treated mice. A, MRTX849 (100 mg/kg), rat IgG2a isotype control antibody (10 mg/kg, Bio X cell lot No. 686318F1B), and mouse PD-1 antibody (10 mg/kg, Bio X cell clone 29F.1A12) were administered orally, daily (MRTX849) and intraperitoneally, every 3 days × three doses (PD-1 and isotype control) alone or in combination as denoted to mice bearing established, subcutaneous CT26 Kras^G12C tumors on the right flank (n = 10/treatment group, average starting tumor volume of ~220 mm³). Vehicle was dosed through study day 10, MRTX849 was dosed through study day 25 and PD-1 was dosed on study days 1, 4, and 7. Data are illustrated as individual tumor volumes over the measured time course for the efficacy and rechallenge studies. B, Survival in the combination-treated cohort was statistically significant compared with all other treatment cohorts by the Mantel-Cox test. “*” denotes adjusted P < 0.05. C, Data in the rechallenge graph depict individual tumor volumes from reimplantation of 1e6 CT26 Kras^G12C cells into the left flank in the one MRTX849-treated mouse and the six combination-treated mice each of which exhibited durable complete tumor responses (CR) in the first implant/study (A). A cohort of naïve mice were also implanted as a control and tumors developed normally in 13 of 14 mice (denoted in purple).

Figure 5.

MRTX1257 reconditions the tumor immune microenvironment and MRTX849 plus anti–PD-1 therapy prolongs survival in Kras^G12C-mutant GEMMs. A and B, Lung tumor-bearing LSL-Kras^G12C Trp53^R270H GEM mice were administered vehicle plus isotype control, vehicle plus anti-mouse PD-1 antibody (10 mg/kg three times per week, Bio X cell clone 29F.1A12), MRTX1257 (50 mg/kg) or MRTX1257 plus anti-mouse PD-1 for 6 days. Mice were euthanized on day 7 and lung tumor nodules were harvested and disaggregated for immune profiling for lymphoid (A) or myeloid (B) populations by flow cytometry. Flow cytometry data are expressed for individual tumors using scatter plots along with mean ± SD for selected cell markers as defined in the Supplementary Materials and Methods. C, Tumor volume was monitored in repeat dose efficacy studies in the LSL-Kras^G12C Msh2^fl/f lung GEM model at baseline and at week 2 and week 4. See Supplementary Fig. S7 for statistical analysis. D, PFS analysis demonstrates combination treatment led to a statistically significant improvement in survival compare with MRTX849 or anti–PD-1 single-agent treatment using the log-rank (Mantel-Cox) test (P < 0.05).